Bottom Line:
Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele.These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families.Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.

ABSTRACTBarley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.

pone-0026468-g001: Construction of Agrobacterium mediated BSMV VIGS vectors with LIC cloning sites and comparisons of BSMV accumulation in N. benthamiana tissues infected with wild type BSMV and its three LIC derivatives.(A) Schematic representation of pCaBS-α, pCaBS-β and pCaBS-γ. Agrobacterium VIGS plasmids were engineered by inserting the respective BSMV α, β and γ cDNAs between the double CaMV 35S promoter and a ribozyme sequence (Rz) derived from TRSV satellite RNA. (B) The organization of three LIC derivatives derived from pCaBS-γ. The LIC site was inserted into pCaBS-γ at three positions downstream of the γa gene. The pCa-γbLIC plasmid was designed to express a full-length γb protein containing two C-terminal amino acid (Glu and Val) extensions preceding the VIGS target sequence. For pCa-LICγb, the LIC site was engineered to replace the AUG start codon of the γb gene and eliminate γb protein expression, and in the case of pCa-LICΔγb, the LIC site was substituted for the γb gene. The primer pairs BS-10 and BS-32 (Table S1), whose positions are indicated above pCa-γbLIC, were used for PCR amplifications to assess the stability of fragments inserted into the pCa-γbLIC vectors. (C) Western blot analysis of BSMV infections after agroinfiltration of N. benthamiana leaves. Analyses of infiltrated leaves are shown at 6 dpi to illustrate virus accumulation before spread to the vascular system. Upper uninfiltrated leaves and roots were assessed at 15 dpi to compare the accumulation of BSMV pCaBS-γ and its three LIC derivatives. Protein was isolated from two plants agroinfiltrated with pCaBS-α and pCaBS-β mixtures containing pCaBS-γ (wt-γb), pCa-γbLIC (γbLIC), pCa-LICγb (LICγb) or pCa-LICΔγb (LICΔγb). BSMV coat protein was detected with BSMV antisera and healthy N. benthamiana leaves (H) were used as negative controls. The bottom panel shows a loading control consisting of Coomassie Blue stained Rubisco, the major protein associated with tobacco leaf extracts.

Mentions:
The three BSMV ND18 cDNAs [64] were cloned into the pCass4-Rz T-DNA vector under the control of a double 35S promoter [58] to permit precise in vivo transcription at the 5′ terminus of the gRNAs. The cDNA α clone was inserted into the StuI site of pCass4-Rz, and the cDNAβ and γ clones were integrated between StuI and BamHI sites to produce pCaBS-α, pCaBS-β and pCaBS-γ (Fig. 1A). The TRSV satellite RNA ribozyme mediated cis-cleavages extending 33, 17 and 17 nts non-viral sequences beyond the 3′ ends of RNAα, β and γ respectively.

pone-0026468-g001: Construction of Agrobacterium mediated BSMV VIGS vectors with LIC cloning sites and comparisons of BSMV accumulation in N. benthamiana tissues infected with wild type BSMV and its three LIC derivatives.(A) Schematic representation of pCaBS-α, pCaBS-β and pCaBS-γ. Agrobacterium VIGS plasmids were engineered by inserting the respective BSMV α, β and γ cDNAs between the double CaMV 35S promoter and a ribozyme sequence (Rz) derived from TRSV satellite RNA. (B) The organization of three LIC derivatives derived from pCaBS-γ. The LIC site was inserted into pCaBS-γ at three positions downstream of the γa gene. The pCa-γbLIC plasmid was designed to express a full-length γb protein containing two C-terminal amino acid (Glu and Val) extensions preceding the VIGS target sequence. For pCa-LICγb, the LIC site was engineered to replace the AUG start codon of the γb gene and eliminate γb protein expression, and in the case of pCa-LICΔγb, the LIC site was substituted for the γb gene. The primer pairs BS-10 and BS-32 (Table S1), whose positions are indicated above pCa-γbLIC, were used for PCR amplifications to assess the stability of fragments inserted into the pCa-γbLIC vectors. (C) Western blot analysis of BSMV infections after agroinfiltration of N. benthamiana leaves. Analyses of infiltrated leaves are shown at 6 dpi to illustrate virus accumulation before spread to the vascular system. Upper uninfiltrated leaves and roots were assessed at 15 dpi to compare the accumulation of BSMV pCaBS-γ and its three LIC derivatives. Protein was isolated from two plants agroinfiltrated with pCaBS-α and pCaBS-β mixtures containing pCaBS-γ (wt-γb), pCa-γbLIC (γbLIC), pCa-LICγb (LICγb) or pCa-LICΔγb (LICΔγb). BSMV coat protein was detected with BSMV antisera and healthy N. benthamiana leaves (H) were used as negative controls. The bottom panel shows a loading control consisting of Coomassie Blue stained Rubisco, the major protein associated with tobacco leaf extracts.

Mentions:
The three BSMV ND18 cDNAs [64] were cloned into the pCass4-Rz T-DNA vector under the control of a double 35S promoter [58] to permit precise in vivo transcription at the 5′ terminus of the gRNAs. The cDNA α clone was inserted into the StuI site of pCass4-Rz, and the cDNAβ and γ clones were integrated between StuI and BamHI sites to produce pCaBS-α, pCaBS-β and pCaBS-γ (Fig. 1A). The TRSV satellite RNA ribozyme mediated cis-cleavages extending 33, 17 and 17 nts non-viral sequences beyond the 3′ ends of RNAα, β and γ respectively.

Bottom Line:
Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele.These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families.Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.

ABSTRACTBarley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.